S.Afr.1.Bot., 1991, 57(6): 325 - 330 325 Pollen morphology and its taxonomic value in () in southern Africa

S.S. Cilliers Department of Sciences, Potchefstroom University for CHE, Potchefstroom, 2520 Republic of South Africa

Accepted 21 August 1991

The pollen morphology of all the southern African representatives of Brachylaena R. Sr. was investigated using light and scanning electron microscopy. On the basis of pollen morphology it is possible to recognize two groups in the . B. discolor (including B. unifloral. B. elliptica, B. glabra, B. neriifolia and B. rotundata are included in Group I, while B. huillensis and B. ilicifolia are the only representatives of Group II. The differ­ ences between the groups primarily concern length and width of colpi, shape of colpus ends, length and width of spinulae, frequency of spinulae, finer tectum sculpture and exine stratification. The possible taxo­ nomic significance of pollen morphology is briefly discussed.

Die stuifmeelmorfologie van al die suider-Afrikaanse verteenwoordigers van Brachylaena R. Br. is met behulp van lig- en aftas-elektronmikroskopie ondersoek. Op grond van stuifmeelmorfologie kan twee groepe binne die genus onderskei word. B. discolor (insluitend B. unifloral. B. elliptica, B. glabra, B. neriifolia en B. rotun­ data ressorteer in Groep I, terwyl B. huillensis en B. ilicifolia die enigste verteenwoordigers van Groep II is . Die belangrikste verskille tussen die twee groepe is ten opsigte van lengte en breedte van kolpusse, vorm van die kolpuspunte, lengte en breedte van spinulas, frekwensie van spinulas, fyner skulptuur van die tektum en eksienstratifikasie. Die moontlike taksonomiese waarde van stuifmeelmorfologie word kortliks bespreek.

Keywords: Asteraceae, Brachylaena, pollen morphology.

Introduction species. The sexine of the pollen of this type Thorough palynological studies on certain representatives of is 1.6 J1.m thick and consists of two layers of baculae (Leins, the Asteraceae have been carried out by various authors 1971). The nexine is 0.8 - 1 J1.m thick and consists of two [Wodehouse (1935) on external morphology and Stix (1960) parts (nexine 1 and 2). He also mentioned that these pollen on internal morphology] . These studies usually concentrated grains are tricolporate and possess narrow colpi with pointed on taxonomically useful characters. Since the usefulness of ends, while the tectum is covered with spinulae. The B. huil­ pollen as a taxonomic tool is restricted by the number of lensis pollen type was found in B. huillensis and B. ilicifolia characters that can be observed by means of light micros­ only and the sexine of their grains is 0.7 J1.m thick and copy (LM) (Wodehouse, 1928), it became apparent to tax­ consists of a single bacula layer. The nexine is undifferen­ onomists that additional pollen characters would have to be tiated and is 0.5 J1.m thick. These pollen grains are tri- or obtained by using transmission (rEM) and scanning (SEM) tetracolporate and possess much wider colpi with rounded electron microscopes (Skvarla et al., 1977). ends and the tectum is also covered by spinulae. The Bra­ As early as 1966 Skvarla and Turner reported on TEM chylaena ramif10ra pollen type is unique to this Madagascar studies of pollen of representatives of most of the tribes of species. the Asteraceae, while Clark et al. (1980), Tomb et al. Leins (1971) also questioned the validity of the inclusion (1974) and others did SEM studies on a selection of repre­ of the subtribe in the tribe Inuleae, on the sentatives of the family. In spite of the palynological basis of their pollen morphology. He recommended the clas­ research in the family, relatively few studies on comparative sification of this subtribe in the tribe , while pollen morphology of taxa were carried out at infrageneric Skvarla et al. (1977) suggested its inel usion within the tribe level (Keeley & Jones, 1977). Anthemidae, also based on pollen morphology. Brachylaena To date, pollen of Brachylaena R. Br. has been studied by was also placed in three other tribes over the years. Keeley means of LM only (Erdtman 1966; Leins 1971). The pollen and Jansen (1991), however, recognized a new tribe, the of Brachylaena is tricolporate and subprolate with an exine Tarchonantheae, with Brachylaena and Tarchonanthus the stratification similar to that of Tarchonanthus (Erdtman, only members, on the basis of chloroplast DNA variation. 1966). Material from one locality only of each of Brachy­ Unfortunately, Leins (1971) did not study pollen of all the laena discolor, B. rotundata and B. neriifolia were studied southern African species of the genus. He also ignored some by Erdtman (1966). representatives of the taxonomically problematic B. dis­ Based on LO analyses of the pollen of most of the mem­ color-unif1ora species complex and in five cases he based bers of the tribe Inuleae, Leins (1971) distinguished three his results on pollen from only one specimen. pollen types in the subtribe Tarchonantheae (to which Bra­ To determine whether pollen morphological characters chylaena belongs), mainly on the basis of exine stratifica­ have taxonomic value amongst the southern African repre­ tion. The Tarchonanthus camphoratus pollen type was sentatives of Brachylaena, it is essential to gain information found by Leins (1971) in four Brachylaena species (B. dis­ from specimens of all the taxa collected from various color, B. rotundata, B. neriifolia and B. elliptica) and four localities. The aim of this study was to search for additional 326 S.-Afr.Tydskr.Plantk., 1991, 57(6)

taxonomically useful characters by using the SEM. To check tectum sculpture. Initially the pollen grains were cleaned by whether the results of Leins (1971) on exine stratification the acetolysis method of Erdtman (1960). Because of colpar hold true for all the Brachylaena taxa in southern Africa, the damage and the fact that this method is time-consuming, SEM studies were complemented with LM studies. the filter technique of Bredenkamp and Hamilton-Attwell (1988) was subsequently used. Materials and Methods The polar axis (P), equatorial width (E) and the thickness Pollen of all the southern African taxa of Brachylaena of the exine and its parts were measured by means of an LM [B. uniflora is included in B. discolor subsp. transvaalensis, provided with an eye piece micrometer. All measurements (Ci II iers , 1990)] were studied by means of LM and SEM. were based on a minimum of ten pollen grains per speci­ Pollen from herbarium specimens from a minimum of four men. The frequency of spinulae was determined from an localities each were studied (Table 1). average of three counts in an area of 2 f-Lm x 2 f-Lm (4 Data on the following characters were recorded: f-Lm 2) in the mesocolpium, by using SEM prints of similar (a) Polarity, size and symmetry of pollen grains (SEM and magnification. LM). The descriptive terminology used mainly follows Erdtman (b) Shape, as well as the amb: equatorial diameter (SEM (1969) and the attempts at standardization offered by and LM); polar diameter (SEM and LM). Reitsma (1970) and Nilson and Muller (1978). The shape of pollen grains in equatorial view was determined from the (c) Aperture type (SEM and LM). shape classes of Erdtman (1966), while terms proposed by (d) Colpus structure (SEM): length of colpus, in equatorial Faegri and Iverson (1975) were used to describe the shape view; width of colpus, in equatorial view; shape of of pollen grains in polar view. colpus ends, in polar view; surface area of the apo­ colpium. Results (e) Tectum sculpture (SEM): length of spinulae; frequency of spinulae in the mesocolpium; finer tectum sculpture Polarity, size and symmetry of the pollen grains (presence of other structures, except spinulae). The pollen grains of all the taxa are released as monads and (f) Exine stratification (LM). are radially symmetrical and isopolar. The polar axis (P) and Some preparation techniques may influence the size and equatorial width (E) of the pollen grains of B. huillensis shape of the pollen grains of representatives of the Aster­ (P = 30 - 37 f-Lm and E = 26 - 35 f-Lm) are larger than those aceae (Cuzma Velari, 1984) and therefore untreated grains of all the other species (P = 26 - 34 f-Lm and E = 20 - 30 were used. Pollen was obtained from unopened florets, f-Lm) . However, as a result of the overlap that exists in the respective dimensions, it is not possible to distinguish be­ mounted directly onto stubs using a carbon glue (,Leit-C tween them on the basis of these characters only. The length nach Gocke', Neubauer Chemikalien), spuller-coated with of the polar axis of the pollen grains of all the Brachylaena gold/palladium and viewed using a Cambridge Stereoscan taxa studied, is 25 - 50 f-Lm, the grains therefore being 250S SEM. Pollen from the same specimens was also pre­ medium in size (ME). pared for LM studies following the method of Coetzee and Van der Schijff (1979). Although only the shape and size of Shape of pollen grains the pollen grains could be studied from these preparations, the pollen was also cleaned to study col pus structure and The shape of the pollen grains of most of the taxa is subprolate (Figure 1), and occasionally prolate spheroidal. Most of the pollen grains of B. huillensis are prolate Table 1 Specimens used in the pollen morphological spheroidal (Figure 2), but sometimes oblate spheroidal or study subprolate. Since the variation in pollen grain shape is continuous, it is not possible to distinguish B. huillensis Taxon Collector, number and herbarium from other species on the basis of their pollen grain shapes. B. discolor subsp. Cilliers 123, 135 (pUC), Hilliard 1673 (UN), Law The pollen grains of most of the taxa studied appeared discolor 5 (NBG), Taylor 206 (UN), Thode Al532 (NH), Venter 1927 (ZULU). spherical in polar view (Figures 3 and 4), and it is therefore B. discolor subsp. Cilliers 119, 140 (pUC), Gerstner 3013 (NH), not possible to determine whether the grains are angulaper­ Iransvaalensis Keet 1130 (STE), Schweickerdt 1368 (NH), turate or planaperturate. Some of the pollen grains of B. ili­ Strey 8787 (NH). cifolia, however, appear triangular with somewhat rounded B. elliplica Abbott 1349 (NH), Moll 5033 (NH), Pegler 997 comers (Figure 5) and are clearly planaperturate. (BOL), Schlechter 2652 (NBG), White 1172 (GRA). B. glabra Dix 130 (GRA), Fourcade 537 (BOL), Thode A860 Aperture type (NH), Van laarsveld 6881 (NBG), West 267 (BOL). The pollen grains of all the taxa studied are colporate, but B. huillensis Cilliers 104 (pUC), Lang s.n. (BOL), Moll & Strey 3755 (NH), Pooley 871 (NH), Ward 3660 (NH). the grains differ in the number of their compound apertures B. ilicifolia Bayliss 5052 (GRA), Compton 19654 (NBG), (combination of ecto- and endo-apertures). Most of the Dahlstrand 1995 (GRA), Feely 62 (NH), Olivier grains of B. huillensis are tetracolporate (rarely tricolporate) 2445 (GRA). (Figure 6). All the other taxa have tricolporate pollen grains B. neriifolia Bolus s.n. (NH), Cilliers 75 (pUC), Duthie (Figure 7). According to NPC analyses all these tricolporate 540 (BOL), Patterson 1267 (GRA). pollen grains have a numerical combination of 345, while B. roluntiala Botha & Ubbink 1556 (pUC), Cilliers 148 (pUC), the numerical combination of the tetracolporate pollen Galpin 6018 (GRA), Leendertz 235 (GRA), Theron grains of B. huillensis is 445. The ora (endo-aperture) of 1557 (PRU). pollen grains of all the taxa studied is lalongate (Figure 8). S.Afr.J.Bot., 1991, 57(6): 325 - 330 327

1

A B

3 4

5

Figures 1- 5 Light micrographs of pollen grains of Brachylaena species. 1. The equatotial view of an untreated pollen grain of B. rotundata (Theron 1557). Scale ba! = 20 J.lm. 2. The equatorial view of an untreated pollen grain of B. huillensis (Cilliers 104). Scale bar = 20 J.lm. 3. A polar optical section through an untreated tricolporate pollen grain of B. neriifolia (Duthie 540) to show the sexine (A), nexine 1 (B) and nexine 2 (C) . Scale bar = 10 J.lm. 4. A polar optical section through an untreated tetracolporate pollen grain of B. huillensis (Cilliers 104) to show the sexine (D) and nexine (E). Scale bar = 10 J.lm. 5. A polar optical section through a tricolporate pollen grain of B. ilicifolia (Dahlstrand 1995). Scale bar = 10 J.lm.

Colpar structure appear to be syncolpate. The species belonging to this group On the basis of the size and shape of the colpus (ecto­ are B. discolor, B. elliptica, B. glabra, B. neriifolia and B. aperture), two groups can be recognized within the taxa rotundata. The colpi of the pollen grains of representatives studied. Pollen grains of the first group (Figures 7 and 9) of the second group (Figures 6, 10 and 11) are not as well possess well-defined colpi that are 18 - 28 fJ.m long and 0.8 defined and are also shorter (10 - 15 fJ.m) and wider (2 - - 1 fJ.m wide with pointed ends. Because of the length of 2.5 fJ.m) than those of the first group. The ends of these the col pi the surface area of the apocolpi is smaller (14 - 27 colpi are rounded and the surface area of the apocolpi vary fJ.m 2) than that of the other group. The surface area of the between 100 and 138 fJ.m 2 • B. huillensisand B. ilicifoliaare apocolpi is so small in some pollen grains that these grains the only representatives of this group. 328 S.-Afr.Tydskr.Plantk., 1991, 57(6)

Figures 6 - 9 Scanning electron micrographs of pollen grains of Brachylaena species. 6. A polar view of an untreated tetracolporate pollen grain of B. huillensis (Cilliers 104) to show the relative surface area of the apocolpium (F) and the rounded ends of the colpi (G). Scale bar = 10 fLm . 7. A polar view of an untreated tricolporate pollen grain of B. discolor subsp. discolor (Cilliers 135) to show the surface area of the apocolpium (H) and the pointed ends of the colpi (I). Scale bar = 10 fLm. 8. A part of a colpus (J) of a cleaned pollen grain of B. neriifolia (Bolus s.n.) to show an os (K). Scale bar = 4 fLm. 9. The equatorial view of an untreated pollen grain of B. elliptica (White 1172) to show the long, relatively thin colpus (L) and the intine (M) pushed through the os (endo-aperture). Scale bar = 10 fLm.

Tectum sculpture Exine stratification Although the tecta of all the taxa studied possess spinulae, The exine of most taxa studied consists of a sexine (1 - 2 two groups can be distinguished on the basis of length and J..lm thick) and a nexine (0.8 - 1.2 J..lm thick) which can be basal width of the spinulae, the number of spinulae per sur­ divided into a nexine 1 and a nexine 2 (Figure 3). The face area, as well as the finer tectum sculpture (openings nexine part of the exine of B. huillensis and B. ilicifolia present or smooth). differs from that of the other taxa in that two parts cannot be The spinulae of the representatives of the first group (Fig­ distinguished and it is also thinner (0.2 - 0.5 J..lm) (Figures 4 ure 12) are longer (0.5 - 0.8 J..lm) and have a wider base and 5). (0.5 - 0.9 J..lm) than those of the second group, with only 2 - 3 spinulae per surface area. Small openings can also be Discussion observed on the surface of the tectum. Since no sections Additional pollen morphological characters that can be used were prepared from the pollen grains, it was not possible to as taxonomic tools were identified from the SEM studies. determine whether these openings are luminae, or only These include characters such as the surface area of the supratectal in origin. B. discolor, B. elliptica, B. glabra, B. apocolpi, spinule frequency and the finer tectum sculpture. neriifolia and B. rotundata are the representatives of this Although pollen morphological characters cannot be used group. The tecta of the pollen grains of representatives of to distinguish amongst all the Brachylaena taxa, it is the second group (Figure 13) possess shorter spinulae (0.2 - possible to define two groups in the southern African repre­ 0.3 J..lm) with narrower bases (0.2 - 0.4 J..lm) and can be sentatives of the genus, on the basis of pollen morphology. classified as nanospinulae. They were found to have a B. discolor (including B. uniflora), B. elliptica, B. glabra, considerably larger number of spinulae (20 - 30) per surface B. neriifolia and B. rotundata are included in Group I, while area than the first group. Because of this comparatively high B. huillensis and B. ilicifolia are the only representatives of spinulae frequency the tectum surface between the spinulae Group II. The main pollen morphological distinctions was not clearly visible, but appeared to be smooth. This between these two groups are summarized in Table 2. LM pollen grain type was found in B. huillensis and B. ilicifolia. observations correspond with most of those of Leins (1971). S.Afr.J.Bot., 1991, 57(6): 325 - 330 329

Figures 10 - 13 Scanning electron micrographs of pollen grains of Brachylaena species. 10. The polar view of an untreated tricolpo­ rate pollen grain of B. ilicifolia (Dahlstrand 1995) to show the surface area of the apocolpium (N) and the rounded ends of the colpi (0). Scale bar = 10 f.lm. 11. The equatorial view of an untreated pollen grain of B. huillensis (Lang s.n.) to show the short but broad colpus (P). Scale bar = 10 f.lm. 12. The finer wall sculpture of a cleaned pollen grain of B. neriifolia (Cilliers 75) to show the spinulae (Q) and openings on the tectum (R) . The square represents that part in which the spinulae were counted. Scale bar = 2 f.lm. 13. The finer wall sculpture of a cleaned pollen grain of B. huillensis (Cil/iers 104) to show the nanospinulae (S). The square represents that part in which the nanospinulae were counted. Scale bar = 2 f.lm.

Characters such as the type of con florescence, the number of Table 2 Summary of the main external morphological heads per conflorescence and anther length can also be used distinctions between pollen of Group I (B. discolor, B. to distinguish between Groups I and II (CiIliers 1990). elliptica, B. glabra, B. neriifolia and B. rotundata) and Different aperture types may be found in poIlen grains of Group " (B. huillensis and B. ilicifolia) obtained from scanning electron microscope studies B. huillensis and B. ilicifolia. However, they correspond with each other on all the other pollen morphological Character Group I Group II characters, and are therefore included in one group. No Aperture type tricolporate tricolporate or tetra· explanation could be found for the variation in aperture type colporate (tricolporate or tetracolporate) in the poIlen grains of B. Length of colpus 18 - 28 f.lm 10- 15 f.lm huillensis. According to Wodehouse (1935) the number and Width of colpus 0.8 - 1.0 f.lm 2 - 2.5 f.lm arrangement of apertures in poIlen grains are determined by Shape of colpus ends pointed rounded the arrangement of the grains prior to the formation of a Surface area of the 14 - 27 f.lm 2 100 - 138 f.lm 2 tetrad. He claimed that it is possible for poIlen grains with apocolpium Length of supratectal 0.5 - 0.8 f.lm 0.2 - 0.3 f.lm four colpi to develop from four daughter cells which were in processes (spinulae) (nanospinuIae) a rhombic or even a square arrangement, and not in the Width of spinuIae bases 0.5 - 0.9 f.lm 0.2 - 0.4 f.lm usual tetrahedral position. Frequency of spinuIae 2-3 20 - 30 Most of the southern African taxa of Brachylaena (repre­ (in 4 f.lm 2) Finer wall sculpture sentatives of Group I) possess the Tarchonanthus camphor­ openings smooth (no openings) atus pollen type described by Leins (1971). The current study, therefore, shows that these taxa have closer affinities with the genus Tarchonanthus. In contrast, B. huillensis and evident from this investigation that pollen morphological B. ilicifolia can be clearly differentiated from Tarcho­ characters (supported by those reproductive characteristics nanthus on the basis of the col par structure, tectum sculpture mentioned earlier) could possibly be used for an infra­ and exine stratification of their poIlen grains. It is also generic division of the genus Brachylaena. To establish such 330 S.-Afr.Tydskr.Plantk., 1991, 57(6)

a division, pollen morphological information on the Brachy­ FAEGRI, K. & IVERSON, J. 1975. Textbook of pollen analysis. laena taxa from Madagascar and the Mascarene Islands will Blackwell, Oxford. have to be obtained. KEELEY, S.c. & JANSEN, R.K. 1991. Evidence from chloro­ plast DNA for the recognition of a new tribe, the Tarchonan­ Acknowledgements theae, and the tribal placement of Pluchea (Asteraceae). Sysl. BOI.16(1): 173-181. The financial support by CSIR, Pretoria, and the Potchef­ KEELEY, S.C. & JONES, S.B. 1977. Taxonomic implications of stroom University for Chri~tian Higher Education is grate­ external pollen morphology to Vernonia (Compositae) in the fully acknowledged. All the herbaria mentioned are thanked West Indies. Am. I . BOl . 64(5): 576 - 584. for the kind loan of their specimens of Brachylaena. I am LEINS, P. 1971. Pollensystematische Studien an Inuleen. I. also indebted to Mr G.F. Smith for his constructive Tarchonanthinae, Plucheinae, Inulinae, Bupthalminae. BOl. comments. lahrb. 91(1): 91 - 146. NILSSON, S. & MULLER, J. 1978. Recommended palynological References terms and definitions. Grana 17: 55 - 58. BREDENKAMP, C.L. & HAMILTON-AITWELL, V.L. 1988. A REITSMA, T. 1970. Suggestions towards unification of descrip­ filter technique for preparing pollen for scanning electron tive terminology on angiosperm pollen grains. Rev. Palaeobot. microscopy. Pollen Spores 30: 89 - 94. Palyn. 10: 39 - 60. CILUERS, S.S. 1990. Taksonomiese ondersoek van die Suider­ SKVARLA, J.J. & TURNER, B.L. 1966. Systematic implications Afrikaanse verteenwoordigers van die genus Brachylaena R. from electron microscope studies of Compositae pollen, a Br. (Asteraceae). M.Sc thesis, Potchefstroom University for review. Ann. Miss. Bot. Gdn., 53: 220 - 244. CHE. SKVARLA, J.J., TURNER, B.L., PATEL, V.C. & TOMB, A.S. CLARK, W.D., BROWN, G.K: & MAYES, R.A. 1980. Pollen 1977. Pollen morphology in the Compo sitae and in morphol­ morphology of Haplopappus and related genera (Compo sitae: ogically related families. In: The biology and chemistry of the Astereae). Am. I. BOl. 67(9): 1391 - 1393. Compositae, eds. Heywood, V.H., Harborne, J.B. & Turner, COETZEE, J. & VAN DER SCHIJFF, H.P. 1979. Pollen B.L., pp. 91 - 110. Academic Press, London. morphology of South African Malvales: Characteristics useful STIX, E. 1960. Pollenmorphologische Untersuchungen an Com­ for keying and for numerical analysis. II. S. Afr. BOl. 45: 93 - 126. positae. Grana Palyn. 2: 41 - 114. CUZMA VELARI, T. 1984. Effects of preparation techniques on TOMB, A.S., LARSON, D.A. & SKVARLA, JJ. 1974. Pollen pollen grains of Cenlaurea weldeniana (Asteraceae). Grana morphology and detailed structure of family Compositae, tribe 23: 91 - 95. Cichorieae, I. Subtribe Stephanomeriinae. Am. I. Bot. 61(5): ERDTMAN, G. 1960. The acetolysis method. Svensk bOI . Tidskr., 486 - 498. 54: 561 - 564. WODEHOUSE, R.P. 1928. Pollen grains in the identification and ERDTMAN, G. 1966. Pollen morphology and plant , classification of , II. . Bull. Torrey Bot. Club Vol. I, 2nd ed. Hafner, New York. 55 : 449 - 462. ERDTMAN, G. 1969. Handbook of palynology. Munksgaard, WODEHOUSE, R.P. 1935. Pollen grains. McGraw-HilI, New Copenhagen. York.